Cancer is "a disease of genetic instability" (National Cancer Institute Director Richard Klausner, as quoted in Science 273:1329, 1996). The process of carcinogenesis is believed to require multiple genetic and/or epigenetic events which affect patterns of expression, or result in mutational alteration, of genes. Various molecular mechanisms may be responsible for the DNA mutation and genetic instability leading to the development of tumorigenesis. Such molecular mechanisms include DNA damage and mutation, alteration of a cell's ability to repair damaged or mutated DNA, alteration of genes responsible for cell-cycle control mechanisms, and alteration of protooncogenes or tumor suppressor genes. However, additional other unknown mechanisms for genetic instability also exist.
DNA damage can result from spontaneous alteration of the DNA molecule, such as a mutation caused during DNA replication, DNA repair, gene rearrangement, or chemical alteration as a result of oxidation or methylation. DNA damage can also result from interaction between the DNA molecule with physical agents (e.g., ionizing radiation); and with chemical agents (e.g., cross-linking agents). A individual gene's degree of sensitivity to DNA damaging agents depends on factors such as the sequence of the gene, and whether it is being actively transcribed or replicated.
There are several pathways by which DNA damage may be repaired (e.g., nucleotide excision repair, enzymatic reversal repair, and postreplication repair). Defects in DNA repair mechanisms may include either somatic or germline mutation of genes encoding proteins necessary for DNA repair and maintenance of genome stability. A defect in DNA repair may accelerate the accumulation of mutations in genes critical to maintain homeostasis, contributing to genomic instability and malignant transformation.
Proliferation of normal cells is associated with protooncogene expression, whereas tumor suppressor genes function in the negative regulation of cell proliferation. Mutations of cellular protooncogenes and tumor suppressor genes could fix a cell in a state of uncontrolled proliferation that could contribute to the development of genomic instability by allowing for replication of damaged DNA before a DNA repair process is complete. Accumulation of a critical number of DNA mutations (genomic instability) can ultimately result in neoplastic transformation.
Genomic instability is believed to occur in an early step in the process of carcinogenesis. Phenotypic changes, directly or indirectly resulting from genomic instability, observed in the progression during neoplastic transformation are used in the diagnosis, staging, and prognosis of malignancies. Thus, a means for detecting and quantitating genomic instability has applications in the diagnosis, staging, and monitoring of cancer patients. Further, therapeutic choices, modalities, and strategies may depend on the accurate assessment of the stage of malignant disease. There are several different manifestations or patterns of genomic instability which have been used successfully in examination of human tumors: abnormal karyotype; increased copy number of genes or of specific nucleotide sequences affecting gene expression; and microsatellite instability.
Microsatellite instability thus represents only one specific manifestation of genomic instability. Microsatellite instability is represented by an alteration in size of microsatellite (simple repeat) sequences. A finding of microsatellite instability represents mutations in one or more genes involved in DNA mismatch repair mechanisms. Thus, while methods for detecting microsatellite instability are important tools in genetic testing of some tumors (e.g., hereditary nonpolyposis colorectal cancer), such methods do not detect major form(s) of genomic instability characterized by molecular aneuploidy arising from deletions, amplifications, translocations, insertions, recombination, and chemical alteration (See FIGS. 1A-1C). For example, microsatellite analysis only specifically detects a subset of colorectal cancers (Bocker et al., 1996, J. Pathol. 179:15-19); of nonsmall cell lung carcinoma (Wieland et al., 1996, Oncol. Res. 8:1-5), and appears not to be particularly useful for small cell lung carcinoma (Adachi et al., 1995, Genes Chromosomes Cancer 14:301-306), for lymphoid neoplasia (Volpe et al., 1996, Ann. Hematol. 72:67-71) or for bone tumors (Tarkkanen et al., 1996, Br. J. Cancer 74:453-5), suggesting a role for other forms of genomic instability in these tumor types.
Accordingly, there is a need in this art for novel, rapid, relatively inexpensive methods and compositions for molecular screening of tumors in quantitating forms and patterns of genomic instability other than the pattern represented by microsatellite instability.